Scattering of photons in biological imaging is a known factor of degrading image resolution and quality. Angular
Domain Imaging (ADI) is a technique which utilizes the angular distribution of photons to filter out multiple-scattering
photons and accept only photons with small angular deviation from their original trajectories. The advantage of ADI is
that it does not require a high optical quality, coherent, or pulsed source to acquire quality image. Initial experiments
with Spatialfrequency Filter (SFF) ADI on simple liquid scattering test phantom showed good results as it can image
through media with scattering ratio (SR) of 106:1. Previous work with complex 3D aquatic species eliminated scattering
but showed optical interference patterns from the coherent laser sources. With SFF ADI, our target is to image through a
complex 3D scattering structure with multilayer of different refractive indices and scattering coefficient from an
Intralipid-infused polymer/agar, and a small species called Branchiostoma lanceolatum, a lancelet that is 5-8cm long and
~5mm thick. To remove interference, several narrow wavelength-band LEDs were used as illumination sources with one
peaks at 630nm and the other peaks at 415nm. The LEDs are collimated and illuminates the 3D structure/lancelet in a
water-filler container while a SFF removes the scattered photons before the imager. This allows us to reduce the optical
interference and to study the impact of switching from coherent laser source into an incoherent narrow wavelength-band
source. Hence, it allows us to investigate the enhancement of imaging the internal structures using the incoherent narrow
wavelength-band source.
Optical imaging through biological tissue has the significant problems of scattering which degrades the image resolution
and quality. Research has shown that Angular Domain Imaging (ADI) improves image quality by filtering out the
scattered light in the biological tissue images based on the angular direction of photons. The advantage of this technique
is that it is independent of the wavelength, coherent, pulse, or duration compared to OCT or time domain. This allows us
to couple ADI with conventional fluorescence imaging technique. Previous work was creating test media by varying
Intralipid/water concentration to produce different scattering levels. This showed difficulties in producing a consistent
scattering medium in liquid states. Hence, ideally we want a reusable solid medium which has a stable scattering
characteristic. Our target is to investigate fluorescence ADI on skin with cancerous collagen tissue where healthy
collagen fluoresces while the cancerous collagen tissue does not. To mimic the characteristic of skin, a solid scattering
medium over a patterned fluorescence material with non-emitting structures is created. We used a solid agar medium, or
a transparent polymer, infused with Intralipid at different concentrations, as the scattering medium. The solid media with
similar scattering characteristic of skin (μs = 20cm-1, g = 0.85) is placed on top of a fluorescence plastic (415nm
excitation, ≈ 530nm emission) which is patterned by strips of non-emitting structures (200-400μm). Using small
apertures with acceptance angles of 0.171° a distance away from the solid scattering medium, these non-emitting
structures are detectable at shallow scattering tissue depth (1-2mm).
Conventional fluorescence imaging often does not have a mechanism to remove the scattering effect in biological tissue.
We use Angular Domain Imaging (ADI) to improve the detection of smaller structures in fluorescence layer over that
can be provided by existing systems. ADI is a high resolution, ballistic imaging method that utilizes the angular
spectrum of photons to filter multiple-scattered photons and accepts only photons with small angular deviation from their
original trajectory. Advantages of the ADI technique are that it is insensitive to wavelength and the sources are not
required to be high quality, coherent, or pulse, as with OCT or time domain. Our target is to perform fluorescence ADI at
shallow tissue such as skin (≈ 1mm) with a buried collagen layer. To experimentally model shallow tissue with phantoms,
a thin layer of scattering medium with similar scattering characteristic (μs = 200cm-1, g = 0.85) is placed on top
fluorescence plastic (415nm excitation, ≈ 555-585nm emission) which is patterned by strips of non-emitting structures
(200-400μm). Positioning multiple collimated arrays with acceptance angles of 5.71° on top of the scattering medium,
test structures (200μm wide) can be detected at shallow scattering medium thickness (1mm). Monte Carlo simulation
confirms that fluorescence ADI can image structures at shallow tissue depth by using collimator array with modest
filtration angles. Results show micromachined collimator arrays provide both high spatial resolution and angular
filtration on scattered photons.
When imaging through small aquatic creatures, scattered photons produce problems in image quality and resolution.
Angular Domain Imaging (ADI) reduces scattered photons and improves the image quality and resolution. ADI is an
imaging technique which utilizes the angular spectrum of photons to filter multiple-scattered photons and accept only
photons with small angular deviation from their original trajectory. Advantages of the ADI technique are that it is
insensitive to wavelength and the sources are not required to be high optical quality, coherent, or pulsed, as with OCT or
time domain. Our target is to image a small species called Branchiostoma lanceolatum, a lancet that is 5-8cm long and
5mm thick, by using ADI to remove the scattering in order to image internal structures. A laser illuminates the lancelet
in a water-filled container and a spatiofrequency filter removes the scattered photons before the imager. Experimentally,
a coherent Nd:Yag second harmonic (533nm) laser creates images but also optical interference occuring within the
internal structures of the lancelet. Conversely, an incoherent
broad-band white light source eliminates the structural
interference effect; however, the wavelength variation of the scattering coefficient combined with the limitation of the
image sensor's dynamic range limit the ability to distinguish the internal structures in many areas. Thus, an IR diode
laser (780nm) is used to lower the scattering coefficient as compared to conventional visible light source and to diminish
the interference effects due to its shorter coherence length.
Researchers have been using simple optics to image optically induced fluorescence in tissues. We now apply the Angular
Domain Imaging technique using a Spatiofrequency filter which accepts only photons within a small deviation angle
from its original trajectory to image a fluorescing medium beneath a scattering layer. A Rhodamine 6 G dye fluorescing
layer or fluorescence slides, under an Intralipid scattering medium was used. By applying ADI with acceptance angle of
0.17°, the structures are distinguishable at low scattering depth depending of the emission wavelength of the
fluorescence source. It was established previously that as the acceptance angle increases, the amount of scattered
light/noise in the images increases, however, the resolution also deteriorates. Simulations using a Monte-Carlo program
are done for both angular filters, Spatiofrequency filter and Linear Collimating Array. Due to the additional positional
filtration on top of the angular filtration with Linear Collimating Array, collimators with aspect ratio as low as 10:1 can
improve the quality of the fluorescence images significantly in both contrast ratio and resolution.
Angular Domain Imaging is an optical tomography technique that filters out scattered light by accepting only photons
with small deviation angles from their original trajectories. Previously, angular filters of linear collimating array (0.29°
acceptance) or spatiofrequency filter of a +50mm lens with a 214um aperture (0.25° acceptance) were used. In the linear
collimating array system, using a wedge prism to deviate the light source by 2-3x the acceptance angle creates a second
image of only the scattered components which can then be subtracted from the filtered image to enhance detectability.
We now apply this technique to the spatiofrequency filter system at an angle 2x the acceptance. Utilizing several
wavelengths of laser sources with different beam symmetries, test phantoms are placed in a 5cm thick sample of diluted
intralipid solution, with a maximum SR of 1.64×106:1 (μs' = 1.8cm-1). By digitally subtracting the background scattered
light, test phantoms previously unobservable are now distinguishable. Using background subtraction, the SR limitation
of the SFF system improves 3x under full illumination and ~40x under line of light illumination. The improvement under
partial illumination is similar to the result using the collimator array, but with resolution limited by the optics used in the
system.
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